Gene transfer in human lymphocytes using retroviral scFv cell targeting

ABSTRACT

The invention relates to gene transfer into human T cells using novel retroviral scFv cell targeting vectors and using these vectors for the treatment of T-cell-associated diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the National Stage of International Application No. PCT/DE00/03444 filed on Sep. 27, 2000, which claims priority from German Patent Application No. 199 46 1422, filed on Sep. 27, 1999, both of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION Background of the Invention

The invention relates to the gene transfer into human lymphocytes, in particular T-lymphocytes using retroviral scFv cell targeting vectors and the use of said vectors for gene therapy, vaccination therapy or diagnostics, in particular for the therapy of T-cell-associated diseases.

The majority of retroviral vectors currently used in gene therapeutic research are derived from the amphotropic murine leukemia virus (MLV). The host cell range of the amphotropic MLV is determined by the surface envelope protein (SU) encoded by the env gene. The protein products of the env gene form the outer envelope of the retroviral vector. The SU proteins interact with i.e. bind to a particular protein (receptor) on the surface of the host cell. The env gene products of the amphotropic MLV allow the gene transfer in a large number of different mammal cells. However, a selective gene transfer into specific cell or tissue types of human or other mammals is not possible with amphotropic MLV vectors since the receptor for the MLV envelope protein on the surface of mammal cells which mediates the entry of amphotropic MLV vectors and the gene transfer may be found on nearly all these cells. Thus, the host cell range of the amphotropic MLV is not specific.

A host cell specificity e.g. is advantageous for the gene therapeutic use, since in a gene therapy outside of the organism (ex vivo) (Anderson et al., 1992; Yu et al., 1997) extensive purifications of the cells are avoided. For the therapeutic, diagnostic or vaccination use in vivo it is desired that retroviral vectors specifically target the desired host cells prior to the transfer of the therapeutic gene. A restriction of the host cell range of the amphotropic MLV could be achieved by modification of the surface envelope protein. A modification of the surface envelope protein was carried out by fusion with a hormone domain. The cells bearing the hormone receptor were transduced (Kasahara et al., 1995). Furthermore, the surface envelope protein has been modified by fusion with a single chain antibody fragment (single chain variable fragments, in the following also referred to as “scFv”). The fragment represented the antigen binding domain of an antibody and is a fusion protein composed of the variable domain Vh and Vl of a monoclonal antibody. The two domains are bound via a glycine and serine oligopeptid [(-(ser-gly4)3-gly-)] enabling the correct folding of the fusion protein (Huston et al, 1991; Whitlow et al., 1991). All modifications carried out heretofore of the MLV surface envelope protein with a scFv show that although the vectors bound to the host target cell no entry into the cell occurred (Russel et al., 1993). Furthermore, it is known that the surface envelope protein of the MLV generally enables no extensive modifications (Cosset et al., 1995). Modifications in which a part of the binding domain of the MLV-SU protein has been replaced often led to an incorrect processing and thus to a defective transport of the SU protein to the cell surface (Weiss et al., 1993; Morgan et al., 1993; Russel et al., 1993). Thus, the development of cell specific retroviral vectors on the base of MLV having altered surface envelope proteins is only less promising.

Retroviral vectors on the base of Spleen Necrosis Virus SNV are more suitable for a targeted gene transfer into e.g. human cells since the surface envelope protein of SNV enables extensive modifications and is also correctly processed (Martinez and Dornburg 1995; Chu and Dornburg, 1994, 1995; Jiang et al., 1998). For the preparation of such vectors at least two components are required. To the one hand, a so-called expression construct has to be prepared which enables a packaging into and the transfer through a retrovirus. The expression construct comprises a coding DNA fragment of the desired gene product, e.g. a gene for gene therapy or as a vaccine. The expression construct has to comprise a nucleotide sequence referred to as packaging signal psi (ψ) which directs the efficient packaging of the mRNA into retroviral particles. Further, a packaging or helper cell is required which provides the gag, pol and env gene products of SNV without packaging the gag, pol and env genes into a retrovirus. The gag, pol and env genes present in the packaging cell have to be psi-negative. After transfer of the expression construct by transfection of the corresponding plasmide DNA into the packaging cells retroviral particles are delivered into the cell culture supernatant, said particles containing the expression construct and being able to transfer only this gene but not the gag, pol and env genes into the target cell. These vectors are unable to propagate and run only through one replication round. The general process for the preparation of propagation unable retroviral vectors is state of the art (Russel et al., 1993, Cosset et al., 1995; Weiss et al., 1993; Morgan et al., 1993; Martinez and Dornburg, 1995; Chu and Dornburg, 1994, 1995; Jiang et al., 1998).

Also the tropism (host cell specificity of the Spleen Necrosis Virus) is determined by the surface envelope protein (SU protein) encoded by the SNV env gene. The SNV surface envelope wild type protein does not permit any selective gene transfer into particular cells or tissues of humans, since the specific recipient protein (receptor) is not present on the surface of human cells (Dornburg, 1995). Therefore, a process has been developed by Dornburg et al., to replace the SNV SU protein for the antigen recognizing domains of antibodies. Said [SNV scFV Env] vectors with four different scFv known heretofore were able to transfer the psi-positive reporter gene, i.e. the bacterial β galactosidase, into selected human target cells (Chu et al., 1994; Chu et al., 1995; Chu and Dornburg, 1997). In detail, there are two scFv expressed against unknown surface antigens on breast and colon carcinoma cells (Chu et al., 1995; Chu and Dornburg, 1997; Jiang et al., 1998), i.e. an scFv directed against the human transferrine receptor and an scFv which recognizes the CD34 surface antigen. A packaging cell line (DSH CXL) has been developed, containing both the psi-negative SNV genes gag, pol and env and the psi-positive reporter-gene (pCXL). Following transfection of the packaging cell with the plasmide DNA of a further expression gene (pTC 53 [expression vector pTC53 and pTC53zeo Jiang et al., 1998]), in which the entire surface envelope protein has been replaced against a single chain antibody fragment (scFv), retroviral vectors were delivered into the cell supernatant which bore in addition to the surface envelope wild-type protein also the chimeric [scFv-Env] surface protein on their surface. By means of said vectors the reporter gene could be transferred into the scFv-specific target cells. In the process described by Dornburg et al., for the preparation of cell specific retroviral vectors it is true that only already known and cloned scFv may be used.

SUMMARY OF THE INVENTION

DE 19752854 A1 describes a method for the preparation of cell type-specific targeting vectors derived from SNV. Up to now, 4 scFv-SNV targeting vectors have been described. They are directed against tumor markers, the transferrine receptor and the CD34 surface antigen (Chu & Dornburg, 1995, 1997, Jiang et al., 1997). Here, the scFv have been derived from monoclonal antibodies (mAb). Furthermore, pseudotype vectors of the type MLV (HIV) for specific transduction of human CD4-positive T cells have been described already (Schnierle & Stitz et al., 1997).

However, no vectors have been described up to now, which are able to transduce human T-cells in a CD4-independent manner.

Thus, an object of the present invention was to provide T-cell specific vectors which are able to transduce T cells in a CD4-independent manner.

The object is solved by cell targeting vectors containing a DNA sequence encoding a single chain antibody fragment (single chain variable fragment, scFv), wherein the single chain antibody fragment has an amino-acid sequence according to any of the FIGS. 1 to 5.

In a preferred embodiment the cell targeting vector further contains a DNA sequence encoding a SNV-env leader according to any of the FIGS. 1 to 5. The cell targeting vectors according to the present invention are T-cell-specific, i.e. the vectors selectively induce human T cells in a CD4 independent manner.

In a further preferred embodiment, the cell targeting vector is derived from SNV (Spleen Necrosis Virus), particularly preferred is the vector pTC53 derived from SNV.

In a further embodiment of the present invention the cell targeting vectors of the invention contain a therapeutic gene. Thus, the invention also relates to the use of the cell targeting vectors of the invention for gene therapy, vaccination therapy or diagnostics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of a nucleic acid sequence of the invention, 7A5-scFv (SEQ ID NO:1), and the amino acid sequence it encodes (SEQ ID NO:6).

FIG. 2 is a representation of a nucleic acid sequence of the invention, K6-scFv (SEQ ID NO:2), and the amino acid sequence it encodes (SEQ ID NO:7).

FIG. 3 is a representation of a nucleic acid sequence of the invention, 7B2-scFv (SEQ ID NO:3), and the amino acid sequence it encodes (SEQ ID NO:8).

FIG. 4 is a representation of a nucleic acid sequence of the invention, 7E4-scFv (SEQ ID NO:4), and the amino acid sequence it encodes (SEQ ID NO:9).

FIG. 5 is a representation of a nucleic acid sequence of the invention, 6C3-scFv (SEQ ID NO:5), and the amino acid sequence it encodes (SEQ ID NO:10).

DETAILED DESCRIPTION

By having the scFv vectors of the invention the first scFv cell targeting vectors are available which are able to transduce human T cells in a CD4-independent manner with a differently high efficiency.

By means of the vectors of the invention, it is now possible to treat following T-cell associated diseases.

(i) Severe Combined Immunodeficiency (SCID). This is a defect in the adenosine-desaminase gene (ada) or the gene encoding thyrosin kinase JAK-3 (Macchi et al., 1995). As a therapeutic Gene the intact ada gene is transferred into T cells by means of the vectors of the present invention. (ii) Acquired Immunodeficiency Syndrome (AIDS) is caused by HIV-1 infection. Therapeutic genes should inhibit the replication or integration of the virus. As therapeutic gene products for intracellular immunization ribozymes, decoy RNA, transdominantly negative mutants of HIV proteins or antibody fragments are suitable (Chang et al., 1994, Ramenzani et al., 1997, Smith et al., 1996, Leavitt et al., 1996, Duan et al., 1995, Levy-Mintz et al., 1996). These therapeutic genes are transferred into the T cells of HIV-1-infected patients by the use according to the invention of the novel cell targeting vectors.

It has been shown that by means of the vectors of the invention (e.g. vectors containing scFv 7A5 shown in FIG. 1; in the following referred to as 7A5 vectors) human macrophages are transduced with a 95% efficiency. Thus, the transfer of therapeutic genes is also possible in HIV-1-infected macrophages by means of said 7A5 vectors.

(iii) T-cell-associated lymphomas.

The (scFv-SNV-Env) targeting vectors of the invention containing a DNA sequence encoding a single chain antibody fragment (single chain variable fragment, scFv), wherein the single chain antibody fragment has an amino-acid sequence (or a fragment) according to any of the FIGS. 1 to 5 selectively enable a transduction of human T-cell lines and partly of primary lymphocytes isolated from blood.

Surprisingly, the vectors of the invention show a selectivity for human T cells which is many times over that for other human cells. The 7-A5-vectors, i.e. the vectors encoding the single chain antibody fragment according to FIG. 1 or a portion thereof, showed a selectivity for human T cells which was increased by a factor of 1000 compared to that for other human cells (c.f. Table 2) and a 4–5 times increased selectivity for T cells compared to B cells.

Table 1 represents 5 scFv (in detail: 7A5, K6, 7B2, 7E4, 6C3) and their vector titers on human T cells (C8166), D17 cells (canine osteosarcoma cell line, permissive for SNV) and HeLa cells (human cervical carcinoma cell line).

Table 2 represents the vector titers of 7A5 vectors. From these data the efficiency and specificity for human T-cells are obvious. By means of said 7A5 vectors T cells which have been made quiescent by gene technologically modified SNV vectors and even human macrophages could be transduced in a very effective manner

Thes following examples illustrate the invention and are not construed to be limiting:

Example 1

Determination of the Vector Titers of 5 Selected scFv on D17, C8166 and HeLa Cells.

For this purpose cell culture supernatants were titered in three serial dilutions (1000 μl, 100 μl and 10 μl) in a total volume of 1000 μl by adding 30 μg/ml polybren on the cells (2×10⁵ D17 and HeLa, 5×10⁵ C8166). After a 1,5-2 h incubation period the vector containing supernatant was replaced by fresh medium.

Following 48 h an X-gal staining was used to detect transduced cells (Mikawa et al., 1992), and the blue cells were counted. Tab. 1 shows the vector titers of the 5 selected scFv on D17, C8166 and HeLa cells.

The titration on D17 (canine osteosarcoma cell line, Watanabe et al, 1983) functions as a positive control for the vector production. The titre of >10⁶ i.U./ml shows that all 5 scFv packaging cell clones deliver vector particles into the cell culture supernatant with about the same efficiency.

The titer on C8166 cells vary between 10³ and 10⁶ i.U./ml depending on scFv, while the transduction on HeLa cells revealed no appreciable titer. Said fact indicates a high selectivity for human T cells of all 5 scFv vectors. The 7A5 vectors most efficiently transduce human T cells (Table 1).

TABLE 1 Vector titers of the 5 scFv vectors. Titer (i.U./ml) ScFv D17 C8166 HeLa 7A5 >10⁶ 1 × 10⁶ <10² K6 >10⁶ 2.5 × 10⁵   <10¹ 7B2 >10⁶ 2 × 10⁴ <10¹ 7E4 >10⁶ 2 × 10³ <10¹ 6C3 >10⁶ 2 × 10³ <10¹

Example 2

Further Characterization of the Vectors

For a detailed characterization, further transduction experiments were carried out with the vectors. In Table 2, the results of the 7A5 vectors are represented.

TABLE 2 Transduction of different cell types by means of 7A5 and wild type vectors Titer (i.U/ml) D17 HeLa TE671 HT1080 293T C8166 Molt4/8 Jurkat A301 huPBMC WT >10⁶ <10¹ <10¹ <10¹ <10¹ <10¹ <10¹ <10¹ <10¹ <10¹ 7A5 >10⁶ <10² <10¹ <10¹ <10² 1 × 10⁵ 1 × 10⁶ 3 × 10⁵ 1 × 10⁵ 7.5 × 10⁴

The transductions were carried out as described above. As a control, all cells were transdued with wild type vectors (WT). These are vector particles only containing the SNV Env wild type protein and no scFv. They are delivered from the starting packaging cell line DSH-cx1 (Chu & Dornburg, 1995, Jiang et al., 1998) into the culture supernatant. As expected, said vectors were not able to transduce human cells. Only the D17 cells which were permissive for them could be transduced with high efficiency.

The titration with 7A5 vectors showed an efficient transduction of several human T cell lines (C8166, Molt4-8, Jurkat, A301), while other human cell types (HeLa: cervical carcinoma, TE671: rhabdomyosarcoma, HT1080; fibrosarcoma, 293T; medulla renalis) could not be transduced. These results show that 7A5 vectors have a high selectivity for T cells.

An increased selectivity for T cells was also found for cell targeting vectors containing a DNA sequence encoding a single chain antibody fragment according to FIGS. 2, 3, 4 or 5.

Example 3

Transduction of Primary T Cells

For the transduction of primary T cells, primary human PBMC (“peripheral blood mononuclear cells”, the isolation of PBMC from blood by means of sucrose density gradient centrifugation is carried out according to standard methods) were isolated from blood.

After a three days stimulation by means of PHA (phytohemaggluttinin) and IL-2 the cell population consisted of 98% T lymphocytes (determined by FACS analysis with an antibody against T cell marker CD3 (state of the art).

The transduction of said cells by means of 7A5 vectors revealed an efficiency of 20% vector positive cells (or approx. 1×10⁵ i.U./ml). As a comparison, the transduction experiments were carried out with human B cells. These could be transduced 5 times less (approx. 4%) than T cells.

Further, stimulated human PBMC could be transduced also with K6 and 7B2 vectors (i.e. vectors encoding the single chain antibody fragment according to FIG. 2 or 3 or a portion thereof). However, this occurred with an efficiency approx. 10 times less than with the 7A5 vectors.

LITERATURE

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1. A cell targeting vector, comprising a DNA sequence encoding a single chain antibody fragment (scFv), characterized in that the single chain antibody fragment comprises amino acid residues 46–329 of SEQ ID NO:6.
 2. The cell targeting vector according to claim 1, further comprising a DNA sequence encoding a spleen necrosis virus (SNV) env leader comprising amino acid residues 1–45 of SEQ ID NO:6.
 3. The cell targeting vector of claim 1, wherein the vector is T cell specific.
 4. A cell targeting vector comprising (a) a DNA sequence encoding a single chain antibody fragment (scFV) comprising amino acid residues 46–329 of SEQ ID NO:6 and (b) a therapeutic gene, wherein the vector is from SNV. 